POB 521, Hungary (A.B.)As a free radical scavenger, and cofactor, ascorbate (ASC) is a key player in the regulation of cellular redox processes. It is involved in responses to biotic and abiotic stresses and in the control of enzyme activities and metabolic reactions. Cytochromes (Cyts) b561 catalyze ASC-driven trans-membrane electron transport and contribute to ASC-mediated redox reactions in subcellular compartments. Putative Cyts b561 have been identified in Arabidopsis (ecotype Columbia) on the basis of sequence similarity to their mammalian counterparts. However, little is known about the function or subcellular localization of this unique class of membrane proteins. We have expressed one of the putative Arabidopsis Cyt b561 genes (CYBASC1) in yeast and we demonstrate that this protein encodes an ASC-reducible b-type Cyt with absorbance characteristics similar to that of other members of this family. Several lines of independent evidence demonstrate that CYBASC1 is localized at the plant tonoplast (TO). Isoform-specific antibodies against CYBASC1 indicate that this protein cosediments with the TO marker on sucrose gradients. Moreover, CYBASC1 is strongly enriched in TO-enriched membrane fractions, and TO fractions contain an ASC-reducible b-type Cyt with ␣-band absorbance maximum near 561 nm. The TO ASC-reducible Cyt has a high specific activity, suggesting that it is a major constituent of this membrane. These results provide evidence for the presence of trans-membrane redox components in this membrane type, and they suggest the coupling of cytoplasmic and vacuolar metabolic reactions through ASC-mediated redox activity.Ascorbate (ASC) plays a key role in the control of growth, development, and defense responses (Davey et al., 2000; Arrigoni and De Tullio, 2002; Mittler, 2002; Pastori et al., 2003). Its role is implied in such diverse processes as the control of cell division and expansion, regulation of programmed cell death, and the regulation of the biosynthesis of ethylene and gibberellic acid. ASC biosynthetic pathways are well characterized in animals and are gradually being elucidated in plants and fungi (Smirnoff, 2001; Smirnoff et al., 2001). In contrast, the cellular mechanisms of ASC regeneration and breakdown remain poorly understood, in particular at the organelle level. The importance of ASC recycling is illustrated by the overexpression of a dehydroascorbate reductase in maize (Zea mays) and tobacco (Nicotiana tabacum), resulting in 2-to 4-fold increased levels of ASC (Chen et al., 2003).A class of membrane proteins, cytochromes b561 (Cyts b561), in plant and animal cells catalyzes transmembrane electron transfer with ASC as the electron donor, thereby contributing to ASC-mediated redox metabolism (Njus and Kelley, 1993; Asard et al., 2001). Members of this protein family use monodehydroascorbate as an electron acceptor, thereby regenerating fully reduced ASC. For example, the chromaffin granule Cyt b561 in the adrenal gland mediates intravesicular ASC regeneration, supporting the biosynt...
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